Long spike twitch-grass E chromosomal RGA SCAR specific molecular label and application thereof

A technology of wheat grass and molecular marker, which is applied in the fields of biological genetic engineering technology and genetic breeding

Inactive Publication Date: 2007-07-18
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, although the candidate gene method using the homologous sequence of the disease resistance gene has isolated a considerable RGAs fragment f...

Method used

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  • Long spike twitch-grass E chromosomal RGA SCAR specific molecular label and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0158] 1. Genomic DNA extraction

[0159] Genomic DNA was extracted by CTAB method. Take 2g of fresh young leaves, grind them into fine powder with liquid nitrogen, add 2×CTAB extract (2% CTAB, 1.4M NaCl, 0.1 MTris-HCl (pH8.0), 0.1M EDTA (pH8.0) preheated to 65°C .0), add 15ml of 2% β-mercaptoethanol before use, and mix well.

[0160] 65 ℃ water bath for 30-45min, during which gently shake to mix.

[0161] After cooling to room temperature, add an equal volume of chloroform:isoamyl alcohol (24:1), mix gently until the supernatant becomes milky, and centrifuge at 4000rpm for 10min.

[0162] Take the supernatant, add an equal volume of isopropanol, and place in an ice bath to precipitate DNA.

[0163] The DNA was hooked out, washed twice with 70% alcohol and once with absolute ethanol, air-dried the DNA, and dissolved in an appropriate amount of TE solution with pH 8.0.

[0164] 0.8% agarose gel electrophoresis was used to detect the DNA concentration and quality.

[0165] ...

Embodiment 2

[0224] 1, with the first step of embodiment 1

[0225] 2. Primer design

[0226] According to the sequence alignment results of DNAMAN5.0 software, degenerate primers were designed at the upstream and downstream of the conserved coding regions of NBS and LRR, respectively. The specific properties of the degenerate primer are as follows:

[0227] RGA-F2: 5'-CGCAACCACTAGAGTAAC-3'

[0228] RGA-R2: 5'-ACACTGGTCCATGAGGTT-3'

[0229] 3, with the 3rd step of embodiment 1

[0230] 4, with the 4th step of embodiment 1

[0231] 4.1 Step 4.1 with Embodiment 1

[0232] 4.2 Step 4.2 with Embodiment 1

[0233] 4.3 Step 4.3 with Embodiment 1

[0234] 4.4 Same as step 4.4 of embodiment 1

[0235] 4.5 Design of specific primers for Chromosome 2E of E. elongatum

[0236] Classify according to the RGAs fragments from different E chromosome sources, and then compare the obtained sequences according to DNAMAN5.0 software, and specifically amplify the specificity of this site at the first ...

Embodiment 3

[0242] 1, with the first step of embodiment 1

[0243] 2. Primer design

[0244] According to the sequence alignment results of DNAMAN5.0 software, degenerate primers were designed at the upstream and downstream of the conserved coding regions of NBS and LRR, respectively. The specific properties of the degenerate primer are as follows:

[0245] RGA-F3: 5'-ATGGGAAGCAAGTATTCAAGGC-3'

[0246] RGA-R3: 5'-TTGGCACAAAATTCTCATCAAGC-3'

[0247] 3, with the 3rd step of embodiment 1

[0248] 4, with the 4th step of embodiment 1

[0249] 4.1 Step 4.1 with Embodiment 1

[0250] 4.2 Step 4.2 with Embodiment 1

[0251] 4.3 Step 4.3 with Embodiment 1

[0252] 4.4 Same as step 4.4 of embodiment 1

[0253] 4.5 Design of specific primers for Chromosome 3E of E. elongatum

[0254] Classify according to the RGAs fragments from different E chromosomes, and then compare the obtained sequences according to DNAMAN5.0 software, and specifically amplify the specificity of this site at the firs...

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Abstract

The present invention discloses specific molecular marking process of E chromosome in long spike couch grass with RGA-SCAR primer. The process includes designing degenerate primers in the upstream and downstream of NBS and LRR conservation coding region, PCR amplification to obtain resistant gene analogy segments on different E chromosomes, designing E chromosome RGA-SCAR specifying primers in NBS and LRR conservation region, and determining the different PCR amplification programs and reaction systems for different primers. The present invention makes it possible to identify precisely different chromosomes of diploid long spike couch grass fast, and provides the foundation of auxiliary molecular selection for improving wheat variety with the exogenous gene of long spike couch grass.

Description

technical field [0001] The invention relates to the fields of biogenetic engineering technology and genetic breeding, in particular to a method for developing E-chromosome-specific primers and novel molecular markers for E. elongatum, and the application of the molecular marker technology in improving wheat genetics and breeding. Background technique [0002] Elongatum elongatum (Host) Beauv.=Elytrigia elongate (Host) Nevski=Thinopyrum elongatum (Host) D.R.Dewey] is an important relative of wheat, with high protein content, resistance to various fungal and viral diseases, It is one of the most widely used wild species in wheat variety improvement due to its excellent traits such as drought resistance, cold resistance, large spikes and multiple flowers. In nature, E. elongatum has three ploidy types: diploid, tetraploid and decaploid. Among them, the E (or E°) contained in the diploid Lophopyrume longatum (Host) A.Lve EE, 2n=14 is considered to be the basic chromosome group...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 郑有良陈国跃魏育明颜泽洪舒晓霞
Owner SICHUAN AGRI UNIV
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